Marine toxin domoic acid alters nitrogen cycling in sediments

As a red tide algal toxin with intense neurotoxicity distributed worldwide, domoic acid (DA) has attracted increasing concerns. In this work, the integrative analysis of metagenome and metabolome are applied to investigate the impact of DA on nitrogen cycling in coastal sediments. Here we show that DA can act as a stressor to induce the variation of nitrogen (N) cycling by altering the abundance of functional genes and electron supply. Moreover, microecology theory revealed that DA can increase the role of deterministic assembly in microbial dynamic succession, resulting in the shift of niches and, ultimately, the alteration in N cycling. Notably, denitrification and Anammox, the important process for sediment N removal, are markedly limited by DA. Also, variation of N cycling implies the modification in cycles of other associated elements. Overall, DA is capable of ecosystem-level effects, which require further evaluation of its potential cascading effects.


Table of Contents
Sediment DA extractions were conducted according to Sekula-Wood et al. 1 .Briefly, 30 mL of 50% MeOH were added to 5 g sediment wet weight, samples were left on a shaker table at 4 ℃ for 12-24 hours in the dark, centrifuged at 3800 RPM for 10 minutes, and filtered through a 0.2 μm filter.DA was quantified on an LC-MS/MS according to a modified protocol using 13 C3-caffeine as an internal standard.
Minimum detection limit for these samples ranged from 0.10-0.15ng/g depending on the analytical run.

Text S2. Rate measurements of potential N transformation rates.
Sediment slurry incubation experiments, with 15 NO3 − (K 15 NO3, purity ≥ 98.5%, Purchased from aladdin) as a tracer, were conducted after 10 and 25 days incubation time, with exetainer tubes for each treatment replicate (n = 5 per treatment) following Seeley, et al. 2 , In brief, exitainer tubes with 2 g of homogenized sediment were helium-purged and dark-incubated overnight to remove residual NO2 − and NO3 − .Six replicates of exetainer tubes (12 mL) per sample were amended with 100 mmol 15 NO3 − (8 mL) and then incubated under experimental conditions.Both anammox and denitrification activities were stopped by adding saturated zinc chloride (ZnCl) solution after 0, 1, and 2 h of incubations.Time series production of 29 N2 and 30 N2 was measured on an isotope ratio mass spectrometer and used to calculate the rate of denitrification and anammox following Jiang, et al. 3 . 15NH4 + was determined by a combination of the ammonium oxidation technique and MIMS analysis (OX/MIMS) 4 .The potential N2 production and DNRA rate were calculated according to the following equation:

R=(K×V)÷W
where R (μmol N kg -1 h -1 ) indicates the measured 15 N-based potential N2 production (RN2) or DNRA rates (RDNRA), K is the slope calculated from the concentration of 15 N-N2 or NH4 + versus incubation time, V (L) is the volume of the incubation vial, and W (kg) denotes the dry weight of the sediment.Partitioning of N2 production and DNRA was expressed as %DNRA (%): Potential nitrification rates were determined for 3 replicates from each group, with 2 g of homogenized, centrifuged and supernatant-water-removed sediment 5 .One hundred millilitres of a solution of 1 mM phosphate buffer (0.3 mM KH2PO4 + 0.7 mM K2HPO4) at pH 7.2 and 0.5 mM (NH4)2SO4 was added to 3 g sediments followed by shaking at 150 RPM at 25 ℃ in aerobic flasks.At Tt = 0, a 10 ml aliquot was transferred to a centrifuge tube, five drops of flocculent solution (0.5 M CaCl2 + 0.5 M MgCl2) were added to aliquots before centrifuging for 10 min at 930 g.Five millilitres of clear supernatant were decanted and analysed for NO3 -+ NO2 - concentration.This procedure was repeated after 18 h of incubation (Tt = 18) and PNR were calculated as differences in the concentration of NO3 -+ NO2 -between Tt = 18 and Tt = 0.For water samples, quality parameters such as NH4 + -N, NO3 − -N, and NO2 − -N were determined according to the standard methods 6 .

Text S3. Detection methods for physicochemical properties and heavy metal.
Total organic nitrogen and carbon levels were measured with a Vario EL III Element Analyzer (Elementar, Germany) 7 .Sediment pH was measured using a pH meter (Mettler Toledo Fiveplus FE20, Shanghai, China) after the dried sediment was soaked in 2 M KCl with a volume ratio of 2.5.
Table S1.Relative abundance of N cycling genes of different treatments on day 10 and 25 (AD0.5 = modified with algal organic matter and 0.5 mg/L domoic acid; AOM = modified with algal organic matter only; CK = without any modification).

Figure S8 .
Figure S8.The neutral community model fit of microbial community in different treatments,

Figure S9 .
Figure S9.Relationships between different nitrogen cycling and quorum sensing genes based on

Table S2 .
Relative abundance of resistance genes of different treatments on day 10 and 25 (AD0.5 = modified with algal organic matter and 0.5 mg/L domoic acid; AOM = modified with algal organic matter only; CK = without any modification).

Table S3 .
The information of the co-occurring network nodes in different treatments (AD0.5 = modified with algal organic matter and 0.5 mg/L domoic acid; AOM = modified with algal organic matter only; CK = without any modification).